Copper(I)-nitrene platform for chemoproteomic profiling of methionine

Methionine plays a critical role in various biological and cell regulatory processes, making its chemoproteomic profiling indispensable for exploring its functions and potential in protein therapeutics. Building on the principle of rapid oxidation of methionine, we report Copper(I)-Nitrene Platform for robust, and selective labeling of methionine to generate stable sulfonyl sulfimide conjugates under physiological conditions. We demonstrate the versatility of this platform to label methionine in bioactive peptides, intact proteins (6.5-79.5 kDa), and proteins in complex cell lysate mixtures with varying payloads. We discover ligandable proteins and sites harboring hyperreactive methionine within the human proteome. Furthermore, this has been utilized to profile oxidation-sensitive methionine residues, which might increase our understanding of the protective role of methionine in diseases associated with elevated levels of reactive oxygen species. The Copper(I)-Nitrene Platform allows labeling methionine residues in live cancer cells, observing minimal cytotoxic effects and achieving dose-dependent labeling. Confocal imaging further reveals the spatial distribution of modified proteins within the cell membrane, cytoplasm, and nucleus, underscoring the platform’s potential in profiling the cellular interactome.

Editorial Note: The schematic figures on pages 32 and 33 were created with BioRender.com,released under a Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International license.

Reviewer #1 (Remarks to the Author):
This paper develops a copper-mediated nitrene transfer process for methionine modification, producing N-sulfonyl sulfimide products.The reactivity study is enlightening, and the use of simple chloramine compounds may well make for simpler synthesis compared to hypervalent iodine or oxaziridines as precursors to nitrenoids.The method easily allows incorporation of useful chemical handles.
Tools to interrogate subtle different in reactivity of side chains brought about by local structure are an important fundamental challenge with large implications for chemical biology.the work here adds to the relatively small set of methionine-selective chemistries, and the demonstration of a copper-mediated mechanistic pathway for nitrene transfer to sulfur is significant and could spur significant revision in the way researchers address methionine reactivity challenges.
Although I think this work is a nice achievement that will be useful for the community, I do have some concerns about aspects of the data, the mechanistic discussion, and the lack of a thorough examination of the attributes that separate this method from previous efforts with oxaziridines, which produce quite similar products.I outline questions that should be addressed below: 1. SI p 94-95: The HPLC traces here are quite broad, and the product peak (4.127 min) has nearly identical retention time to the starting material.MS S/N is rather poor, so I'm concerned that quantification is questionable here.Furthermore, what is the unidentified material at retention time ~ 6.5 min?2. The paper give insufficient credit to Chang's prior with nitrene transfer using oxaziridine reagents.Some important precedent is not cited (although the initial report is cited): https://doi.org/10.1021/acscentsci.9b01038(Ohata... Chang) http://dx.doi.org/10.1021/jacs.9b04744(Christian... Chang) 3. A major motivation for this work is "the poor stability of strained oxaziridines and the resulting sulfimide conjugates towards hydrolysis limits its applications in live cells and for making covalent inhibitors."In this regard, the stability test (Fig 3) seems insufficient.The studies here were not performed under identical conditions to the Chang work.A direct stability comparison of the N-sulfonyl sulfimides (Chang work) to the present Naminocarbonyl sulfimides seems necessary, given that the evidence indicates that both are >50% stable after many days.
4. Relative to the Chang work, this effort is plagued by competitive sulfoxidation, lowering yields and producing product mixtures.In that sense, I don't really see how this is a concrete advance on prior art.
5. Care must be taken not to assume too much about the mechanism and role of copper here.Copper nitrene intermediates, although plausible here, are certainly not the only explanation or mechanism.Observation of copper nitrene-like species is extremely limited, and other roles for copper are plausible.Although copper nitrene is a reasonable intermediate to assume, and it certainly provides a motivation to develop the experiments in this direction, computing the structure and HOMO-LUMO gap for a copper nitrene (Fig 3c) feels overly specific given available data.species, but the view of a Cu(II) species here is unusual and should be referenced.This point is also relevant to the computed structures as well.Why have the authors assumed Cu(II)?
The key intermediates here are often termed carbenoids in the literature because the intermediate is indeed largely unknown.
10.Other issues with the mechanism: the bromide atom disappears in the first arrow, raising questions about conservation if charge if a bromide leaving group is assumed.
11.The mechanism draws specific ligands into some structures and not others, and does not use bracket (e.g.[Cu]) notation to indicate unspecified ligands.
12. The MS data for modified lysozyme, chymotrypsinogen A, creatine kinase, BSA seems to be missing from the SI?How are conversions and yields determined here?
13.Some other protein data is of relatively poor quality.This is common for larger proteins with ensemble mixtures of modifications, but care must be taken in quantification in these cases.What do we make of the peak in apo-transferrin modification at 79906, for example?Is this consistent with a double-modification?The authors have developed copper(I)-catalyzed nitrene Platform (CuNiP), which is a highly chemoselective method for methionine labeling to generate highly stable sulfonyl suofimide conjugates.I'm not familiar with the experimental sections, and thus I only discuss the computational section.They have demonstrated CuNiP selectively labeled methionine residues in a wide range of bioactive peptides, intact proteins and proteins in complex cell lysate mixtures.NBO analysis revealed the origin of the stability of sulfonyl sulfimides, and evaluated the effects of substituents on the chloramine-T in the efficiency of methionine labeling.These findings exhibit a potential to improve the understanding of cellular processes and disease pathogenesis.I recommend this manuscript to be published in this journal after addressing the minor issues.
(1) Page 10, the sentence in lines 255-257 "The results from these analysis showed that the electron density on the nitrogen (QN) of the copper-nitrene complex increases when an electron-donating groups are present (1a; QN-Mulliken = -0.230,1b; QN-Mulliken = -0.242)hence the increased partial negative charge (Fig. 4b, Supplementary Fig. 18).".In this passage, the last part "hence the increased partial negative charge" seems incomplete, and thus the authors should reorganize this part.
(2) Page 12, the sentence in lines 310-315."The ability of CuNiP to selectively modify particular methionine sites among several methionine residues on native proteins This preference for labeling a particular methionine site(s) in the presence of multiple methionine residues on proteins is in line with the chemoproteomics principal where hyperreactive Met undergo selective labeling based on the microenvironment thus shows the remarkable ability of this platform to detect hyperreactive Met in a proteome."There are serious grammatical errors in this passage, and thus the auathors should give a revision.

Reviewer #3 (Remarks to the Author):
In this manuscript, Sadu et al. developed a novel methionine probe based on copper(I)nitrene and used it to profile breast and prostate cancer cell lines via LC-MS/MS and microscopy.The authors demonstrated a commendable approach in the development of probe 1a, employing a rational design strategy coupled with Density Functional Theory (DFT) calculations.The investigation is very thorough and the probe works as expected on amino acids, peptides, purified proteins, and more complex systems like cell lysates and living cells.
However, there are a certain number of aspects that need to be addressed before this manuscript becomes publishable in Nat Commun.
• The experiment shown in Figure 3e should be repeated with the same amino acids on the peptide plus a methionine and in the negative control an alanine instead, as to present a yield for the reactions.
• The authors should calculate the selectivity for methionine from the chemoproteomic experiment presented in Figure 8a by searching for the expected adduct on Met, Lys, Gln, Tyr, Cys, Arg, Asp, His, and Ser and report the result as a bar graph.
• The authors explained in Figure 5 that mostly surface-exposed methionine are labeled.Does this still hold true for the 296 identified Met in the lysate experiment in Figure 8a?• The concept of hyperreactive methionine is used to describe the probe labeled methionine; however, no data is presented to support this argument.The only explanation explored in Figure 5 was solvent accessibility, which differs from hypereactivity.The authors should consider removing the statement on hyperreactivity or provide data to support it such as calculating intensity ratios between the different tested concentrations from 1 uM to 250 uM and the sites harboring a lower or no increase in intensity among concentrations would be hyperreactive and worth discussing in more details.In other words, provide evidence that these residues are saturated at lower concentrations of probe labeling, suggesting hyperreactivity.
• As mentioned other methionine reactive probes exist, notably using an oxaziridine warhead (ReACT platform) and this probe usually identifies around 1000 methionine (He et al., 2022, Molecular Cell 82, 3045-3060).Is there any overlap between their identified methionines and the ones identified here?It would add value to this new probe to compare the methionine that can be profiled with these different warheads.
• The fluorescence gel presented in Figure 9b using probe 1i (1a alkyne) for labeling on live cells shows only 2-3 bands around 75 kDa starting at 500 uM.The authors should perform the LC-MS/MS experiment presented in Figure 8a in live cells and report the number of methionine enriched as currently, it seems that this probe labels very few proteins on live cells which is not the purpose of a broad profile probe.
• How many cells were quantified in the microscopy presented in Figure 9c?Only 3 cells are shown and not quantified.The authors should quantify at least 20 cells.Further, the authors claim that the probe labels cytoplasmic and nuclear proteins.However, from the single image, it is impossible to differentiate between cell surface labeling and intracellular labeling.A Z-stack image should be acquired to support this claim.
• This manuscript shows a very methodological and rational development of probe 1a but an application of the probe would greatly strengthen the study.The authors end this work by showing that live cells can be labeled.A suitable example could be to profile oxidationsensitive methionines via the platform shown in Figure 8a.

TITLE: Copper(I)-Nitrene Platform for Chemoproteomic Profiling of Methionine
We want to take this time to sincerely thank the reviewers for their insight into bettering our research.We have addressed the reviewers' concerns in a revised version of our manuscript.Please see our responses to the reviewers below.

Reviewer #1 (Remarks to the Author):
This paper develops a copper-mediated nitrene transfer process for methionine modification, producing N-sulfonyl sulfimide products.The reactivity study is enlightening, and the use of simple chloramine compounds may well make for simpler synthesis compared to hypervalent iodine or oxaziridines as precursors to nitrenoids.The method easily allows incorporation of useful chemical handles.
Tools to interrogate subtle different in reactivity of side chains brought about by local structure are an important fundamental challenge with large implications for chemical biology.the work here adds to the relatively small set of methionine-selective chemistries, and the demonstration of a copper-mediated mechanistic pathway for nitrene transfer to sulfur is significant and could spur significant revision in the way researchers address methionine reactivity challenges.
Although I think this work is a nice achievement that will be useful for the community, I do have some concerns about aspects of the data, the mechanistic discussion, and the lack of a thorough examination of the attributes that separate this method from previous efforts with oxaziridines, which produce quite similar products.I outline questions that should be addressed below: We want to thank the reviewer for these comments and the opportunity to address his/her concerns.We thank them for recognizing the impact of our research.
Comment: SI p 94-95: The HPLC traces here are quite broad, and the product peak (4.127 min) has nearly identical retention time to the starting material.MS S/N is rather poor, so I'm concerned that quantification is questionable here.Furthermore, what is the unidentified material at retention time ~ 6.5 min?

Justification:
We have re-performed the experiment with tetracosactide acetate and alkyne probe 1i. 2 mg of tetracosactide acetate (final conc.1.25 mM) was dissolved in MeCN:H 2 O and to it were added CuBr (110 µL from 20 mM stock solution, final conc.6.25 mM, 5.0 equiv), and 1i (340 µL from 10 mM stock solution, final conc.6.25 mM, 5.0 equiv).The resulting solution was stirred at RT for 3 h and quenched by adding 10 µL 0.5 N HCl.The crude mixture was analysed using HPLC (2-80% solvent B over 30 min, 1 mL/min, solvent B: 0.1% formic acid in MeCN) to determine the conversion and analyzed by MS.This data has been added into the revised supporting information (Supplementary Fig. 30).Comment: A major motivation for this work is "the poor stability of strained oxaziridines and the resulting sulfimide conjugates towards hydrolysis limits its applications in live cells and for making covalent inhibitors."In this regard, the stability test (Fig 3 ) seems insufficient.The studies here were not performed under identical conditions to the Chang work.A direct stability comparison of the N-sulfonyl sulfimides (Chang work) to the present N-aminocarbonyl sulfimides seems necessary, given that the evidence indicates that both are >50% stable after many days.

Justification:
The primary objective of our work is to develop new, mechanistically and structurally different labelling strategies for methionine and apply them for profiling of hypereactive methionine that cannot be captured by current methionine selective probes.In response to your feedback, we have revised the manuscript to emphasize the novel contributions of our Copper(I)-Nitrene Platform (CuNiP) and its utility in chemoproteomic profiling in both vitro and live cells.Our new studies which are highlighted in details below led to the discovery of new protein labeled sites, identification of novel labelling sites and live cells proteomics.We removed the stability comparison with oxaziridine-based sulfimides, focusing instead on the innovative aspects of our work.
Comment: Relative to the Chang work, this effort is plagued by competitive sulfoxidation, lowering yields and producing product mixtures.In that sense, I don't really see how this is a concrete advance on prior art.

Justification:
We acknowledge the reviewer's concern regarding the competitive sulfoxidation observed in 2. Identification of Novel Labeling Sites: Within the subset of proteins that were labeled by both CuNiP and the oxaziridine platform, our method was able to identify 55-63% new protein sites (for low and medium dose) across low-dose, medium-dose, and high-dose.This highlights the ability of CuNiP to label new sites within the same proteins.

Live cell Proteomics:
Further expanding CuNiP, we performed methionine profiling inside live cells, labelling proteins at their native states.Proteomics analysis of live-cells clearly showed a dose-dependent labelling of methionine residues on proteins with 1i (Fig. 9c, revised manuscript; 100 µM = 20 proteins; 250 µM = 42 proteins; 500 µM = 229 proteins; 1 mM = 305 proteins; 2 mM = 236 proteins).Gene ontology (GO) analysis of modified proteins further corroborates the presence of significant membrane modification in live cell samples (Fig. 9c, revised manuscript).Furthermore, functional categorization of modified proteins showed a broad diversity in the classes of proteins modified (Fig. 9d, revised manuscript).This result highlights the application of CuNiP for profiling of methionine sites in proteins in live cells.
We believe that the Copper(I)-Nitrene Platform marks a significant step forward towards methionine bioconjugation as well as chemoproteomic profiling.It's ability to identify new proteins and labeling sites, combined with the stability of its conjugates, positions it as a valuable tool.Different labelling strategies may be complementary and collectively enrich the toolkit available to researchers.CuNiP adds a valuable dimension by offering alternative mechanisms for methionine labeling.We hope that our revised manuscript now clearly conveys the innovative aspects and the significance of our method in the context of the existing body of work.
Comment: Care must be taken not to assume too much about the mechanism and role of copper here.Copper nitrene intermediates, although plausible here, are certainly not the only explanation or mechanism.Observation of copper nitrene-like species is extremely limited, and other roles for copper are plausible.Although copper nitrene is a reasonable intermediate to assume, and it certainly provides a motivation to develop the experiments in this direction, computing the structure and HOMO-LUMO gap for a copper nitrene (Fig 3c) feels overly specific given available data.

Justification:
The formation of nitrene species in the presence of copper catalysts, particularly in the context of reactions involving Chloramine-T has been well documented in literature in Aziridination, sulfimidation, C-H amination etc. (J.Org.Chem.1997, 62, 6512-6518; Journal of Molecular Catalysis A: Chemical, 2002, 85-89;Tetrahedron Letters, 1969, 3301-3304;Tetrahedron Lett. 1997, 38, 7453;Org. Biomol. Chem., 2005, 3, 107-111).Therefore, we believe that the reaction is proceeding via an nitrene/nitrenoid species.Although there is no direct evidence in observing the Cu-nitrene species, we observed that in presence of Cu-salts, chlorination of K, Y, N-terminus is totally supressed, thus further supporting the presence of Cu-nitrene species.Therefore, we believe it is plausibly going via nitrene mechanism.

Comment:
The computed HOMO-LUMO gap of 5.4 eV = 124 kcal/mol: is this a reasonable number for a fast room temp reaction?

Justification:
We have re-evaluated our HOMO-LUMO calculations, and the observed gap of 5.4 eV is consistent.A similar trend is observed for the computational analysis of oxaziridine probe with methionine which had a HOMO-LUMO gap of 7.4 eV (170 kcal/mol) as reported in (J. Org. Chem. 2017, 82, 9765-9772).Furthermore, evaluation of the HOMO-LUMO gap of oxaziridine using density functional theory method B3LYP-D3(BJ), 6-311g++(d,p) basis set, as demonstrated in our manuscript, gave a gap of 5.07 eV (116 kcal/mol).Consequently, we would like to state that the HOMO-LUMO gap only addresses relative reactivity based on frontier orbital gap of optimized low energy conformer geometries of methionine and Cu-Nitrene-Ligand complex.Reactivity of methionine with Cu-Nitrene-Ligand complex is far more complex and requires geometry optimization of interacting species, favourable orientation of reacting species in space, binding energy, enthalpic and enthropic changes.This can be achieved by calculating the full mechanistic pathway for methionine and other reactive residues, and this is beyond the scope of the current manuscript.Work in this direction is currently going on in our lab.

Comment:
The way the study on variation in sulfonyl structure (

Justfication:
In the revised manuscript, we have redesigned the experiment to understand the role of electron-donating vs electron-withdrawing group in the product outcome of sulfonyl sulfimidation.Since it was not clear to identify the trend on a protein level, we have performed the same on a model pentapeptide Fmoc-VKQMK-CONH 2 under our optimized condition.

MS spectra of 1a modified apo-transferrin
Deconvoluted MS spectra of 1a modified apo-transferrin

MS/MS Analysis of 1a modified apo-transferrin:
Identified Peptide Sequence: LCMGSGLNLCEPNNK (AA-516-AA530)  Response: We fully agree with the reviewer's comment.To assess the selectivity for methionine, we performed a database search with the modification on Met, Lys, Gln, Tyr, Cys, Arg, Asp, His, and Ser.A limited number of modified peptides can be identified when searching for other amino acids at varying probe concentrations.We believe these observed modifications are artifacts as we do not observe any reactivity of CuNiP with serine or other reactive residues at peptide and recombinant protein examples.This has been added in revised supporting information (Supplementary Fig. 34).

Comment:
The authors explained in Figure 5 that mostly surface-exposed methionine are labeled.Does this still hold true for the 296 identified Met in the lysate experiment in Figure 8a?
Justification: Regarding solvent accessibility of 1i modified methionine residues, we have calculated the solvent accessible surface area (SASA) of 208 random peptides from the high dose (250 µM) samples in Fig. 8a.Out of 208 sites tested, 155 sites (74%) were found to be buried whereas 53 sites (26%) were solvent exposed.These results highlight the potential of CuNiP to label methionine residues irrespective of their position on a protein.This has been added to the revised manuscript and supporting information (Supplementary Fig. 34).

Comment:
The concept of hyperreactive methionine is used to describe the probe labeled methionine; however, no data is presented to support this argument.The only explanation explored in Figure 5 was solvent accessibility, which differs from hypereactivity.The authors should consider removing the statement on hyperreactivity or provide data to support it such as calculating intensity ratios between the different tested concentrations from 1 µM to 250 µM and the sites harboring a lower or no increase in intensity among concentrations would be hyperreactive and worth discussing in more details.In other words, provide evidence that these residues are saturated at lower concentrations of probe labeling, suggesting hyperreactivity.
Justification: Based on the valuable comments from the reviewer, we have now incorporated data highlighting hyperreactive peptide clusters for the different concentrations.We have generated a heatmap and peptide cluster map identifying the hyperreactive modified methionine sites, with cluster 11 containing 9 proteins with hyperreactive methionine sites.This has been added in revised manuscript (Fig. 8b) and supporting information (Supplementary Fig. 34).
Comment: As mentioned other methionine reactive probes exist, notably using an oxaziridine warhead (ReACT platform) and this probe usually identifies around 1000 methionine (He et al., 2022, Molecular Cell 82, 3045-3060).Is there any overlap between their identified methionines and the ones identified here?It would add value to this new probe to compare the methionine that can be profiled with these different warheads.8a in live cells and report the number of methionine enriched as currently, it seems that this probe labels very few proteins on live cells which is not the purpose of a broad profile probe.
Response: Looking at the fluorescence gel presented in Figure 9b, we can see why it seems like only 2-3 proteins were modified.We believe that these proteins are abundant proteins as can be seen in the Coomassie gel.However, a close-up look at the fluorescent gel clearly highlights a broad distribution of modified proteins across different molecular weights.Consequently, we also carried out proteomics analysis on the labelled live T47D cells and observed a similar dose dependent labelling (20 proteins-100 µM, 62 proteins-250 µM, 229 protein-500 µM, 305 proteins-1 mM, 236 proteins-2 mM).We attribute the lower number of proteins observed for 2 mM to be associated with increasing cell death as concentration of CuNiP reagent increases.Interestingly, GO analysis of these proteins clearly identified a broad range of function and localization of modified proteins within the cell, with a significant number of modified proteins been cell membrane-related proteins.This has been added in revised manuscript (Fig. 9b) and supporting information (Supplementary Fig. 37).
Comment: How many cells were quantified in the microscopy presented in Figure 9c?Only 3 cells are shown and not quantified.The authors should quantify at least 20 cells.Further, the authors claim that the probe labels cytoplasmic and nuclear proteins.However, from the single image, it is impossible to differentiate between cell surface labeling and intracellular labeling.A Z-stack image should be acquired to support this claim.

Response:
We appreciate the reviewer's suggestion on quantifying more cells and acquiring a z-stack image highlighting the spatiotemporal localization of modification within cells.Consequently, we have repeated the experiment and quantified >50 cells for control and experimental samples.Also, we have included a z-stack image and a gif supplementary material of median intensity image from 22 slices of CuNiP modified cells.From these recent images, we can clearly observe that most of the modifications were at the cell membrane while few modifications were within the nuclear region of the cells.This observation largely concurs with the proteomics results obtained from live cell experiments.This has been added in revised manuscript (Fig. 9e) and supporting information (Supplementary Fig. 38).
Comment: This manuscript shows a very methodological and rational development of probe 1a but an application of the probe would greatly strengthen the study.The authors end this work by showing cells can be labeled.A suitable example could be to profile oxidation-sensitive methionines via the platform shown in Figure 8a.
Justification: We thank the reviewer for suggesting such an exciting application for CuNiP reaction.Consequently, we have successfully probed for oxidation sensitive methionine within the human proteome using CuNiP.This was achieved by treating experimental samples with 0.5-2 mM of hydrogen peroxide for 1 h, followed by labelling with 1i using CuNiP, in-gel fluorescence analysis, and proteomics analysis.Comparison of experimental samples to control sample (without H 2 O 2 treatment) readily shows a decrease in fluorescent intensity, suggesting the ability of CuNiP to modify oxidation sensitive methionine residues.Proteomics analysis further corroborates this observation as we observed a dose-dependent decrease in modified methionine sites as hydrogen peroxide increases (356 peptides-control; 267 peptides-0.5 mM; 178 peptides-1 mM; 115 peptides-2 mM) with (0.5 mM of H 2 O 2 , 88 PSMs < control; 1 mM of H 2 O 2 , 177 PSMs < control; 0.5 mM of H 2 O 2 , 240 PSMs < control).Further analysis led to the discovery of 86 sites that were only modified in control samples but not in any of the H 2 O 2 concentrations.Interestingly, Gene Ontology analysis of these proteins containing oxidation sensitive methionine residues (86 sites) showed significant enrichment of nucleic acid metabolism proteins, thus suggesting a plausible protective role of methionine in gene regulation and cell division.This has been added in the revised manuscript (Fig. 8e-8g) and supporting information (Supplementary Fig. 35).

In-gel fluorescence analysis:
Proteomics analysis:

REVIEWERS' COMMENTS
Reviewer #1 (Remarks to the Author): The revisions have addressed most of the primary concerns raised upon initial review, and publication now seems warranted.

Reviewer #3 (Remarks to the Author):
In this revised manuscript, Sadu et al. addressed all of my issues with the proposed experiments.
As a minor comment, while the observed amino acid selectivity clearly favors labeling of methionine, I would not simply disregard the modification on other amino acids simply on the basis that this was not observed on peptide/purified proteins as depending on the protein microenvironment these may very well be real in a complex system like cellular lysate or live cells.
In conclusion, I now support the publication of this manuscript in Nat Commun.

TITLE: Copper(I)-Nitrene Platform for Chemoproteomic Profiling of Methionine
We want to take this time to sincerely thank the reviewers for their insight into bettering our research.We have addressed the reviewers' concerns in a revised version of our manuscript.Please see our responses to the reviewers below.
Reviewer #1 (Remarks to the Author): Comment: The revisions have addressed most of the primary concerns raised upon initial review, and publication now seems warranted.
Justification: We thank the reviewer for accepting our revisions.
Reviewer #3 (Remarks to the Author): In this revised manuscript, Sadu et al. addressed all of my issues with the proposed experiments.
Comment: As a minor comment, while the observed amino acid selectivity clearly favors labeling of methionine, I would not simply disregard the modification on other amino acids simply on the basis that this was not observed on peptide/purified proteins as depending on the protein microenvironment these may very well be real in a complex system like cellular lysate or live cells.
In conclusion, I now support the publication of this manuscript in Nat Commun.

Justification:
We appreciate your insightful comment and agree with your perspective.We acknowledge that the modification of other amino acids, although not predominant in our peptide/purified protein experiments, could indeed occur under the complex conditions present in cellular lysates or live cells.This possibility will be highlighted as a consideration for future studies and in the interpretation of our results in more heterogeneous environments.Thank you for bringing this important point to our attention.
6.The computed HOMO-LUMO gap of 5.4 eV = 124 kcal/mol: is this a reasonable number for a fast room temp reaction?7. The way the study on variation in sulfonyl structure (Fig 4) is conducted does not really shed light on structure-reactivity relationships.All reagents give >95% conversion, and fairly subtle variation in selectivity.As it stands, I would either delete Fig 4b, or else re-design the reactivity studies to measure kinetic differences and/or conditions with lower conversions.8.The mechanistic scheme (Fig 4b) also has concerns."Hydrolysis" here is unclear and perhaps overly simplistic.Complexation of a thioether group to a copper center should not render the S electrophilic for nucleophilic attack by a water molecule.9. Copper nitrene transfer chemistry is classically understood as a Cu(I) intermediate (e.g.https://doi.org/10.1021/ja00126a044,or https://doi.org/10.1039/D3SC03641C,or https://doi.org/10.1039/10.1002/ijch.201900181), or sometimes formally as a Cu(III) 14. How are the ratios of various Met sites measured (Fig 5).Proteomics data of MS peaks heights after digestion is not typically quantitative.15.Some discussion of Fig 8c may be useful.What software was used in its creation?Is it possible that the underrepresentation of K/R residues is an experimental artifact due to changes in trypsin cleavage induced by local modification?Reviewer #2 (Remarks to the Author): 1i modified Tetracosactide Acetate NH 2 -SYSMEHFRWGKPVGKKRRPVKVYP-OH: LCMS m/z 1570.8037(calc.[M+2H + ] 2+ = 1571.3049),1047.8753(calc.[M+3H + ] 3+ = 1048.8723),m/z 786.1547 (calc.[M+4H + ] 4+ = 786.1553),m/z 629.1276 (calc.[M+5H + ] 5+ = 629.1263)Purity: > 99 % (HPLC analysis at 220 nm).Retention time in HPLC: 6.995 min.HPLC trace of the unmodified tetracosactide acetate MS Spectra of unmodified tetracosactide acetate HPLC trace of the 1i modified tetracosactide acetate (Note: The peak at 4.08 min does not correspond to any peptide fragement, therefore we presume that it is coming from organic impurity) MS spectra of 1i modified tetracosactide acetate MS spectra of unidentified impurity at 4.08 min showing no peptide fragment is present.Comment: The paper give insufficient credit to Chang's prior with nitrene transfer using oxaziridine reagents.Some important precedent is not cited (although the initial report is cited): https://doi.org/10.1021/acscentsci.9b01038(Ohata...Chang) http://dx.doi.org/10.1021/jacs.9b04744(Christian...Chang) Justification: Necessary citation has been added in the revised manuscript.(Ref 16 & 17 in the revised manuscript) Fig 4) is conducted does not really shed light on structure-reactivity relationships.All reagents give >95% conversion, and fairly subtle variation in selectivity.As it stands, I would either delete Fig 4b, or else redesign the reactivity studies to measure kinetic differences and/or conditions with lower conversions.

Justification:
Since the abovementioned report of methionine profiling was carried out on a mouse organoid model, for better comparison, we have compared the protein list labeled by oxaziridine and CuNiP based on the original work by Chang et al. (Science 2017, 355, 597-602).At identical probe concentration (10 µM-low dose, 50 µM-medium dose, 250 µMhigh dose), CuNiP was able to identify 63-78% new proteins that were not labeled by the oxaziridine platform.Interestingly, within the common protein targets under each probe concentration, CuNiP was able to label 55-63% new protein sites (Low & medium doses).This tells us that, due to its unique labeling mechanism and structurally different warhead, CuNiP was able to identify a substantially large set of proteins and protein sites which was not identified before.10 µM (low-dose) 50 µM (medium-dose) 250 µM (high-dose) Comment: The fluorescence gel presented in Figure 9b using probe 1i (1a alkyne) for labeling on live cells shows only 2-3 bands around 75 kDa starting at 500 µM.The authors should perform the LC-MS/MS experiment presented in Figure

At identical probe concentrations (10 µM-low dose, 50 µM-medium dose, and 250 µM-high dose), the CuNiP platform labeled 63-75% new proteins than oxaziridine probe.
The comparisons of new identified labelled methionine sites between CuNiP and Chang's oxaziridine probe have yielded promising results.Although the two cell lines were different (HeLa for oxaziridine and T47D for CuNiP) reports suggest that they share upto 96% proteome similarity (Mol.Cell.Proteomics, 2012, 1-11).This indicates a significant extension of the chemoproteomic landscape, unveiling new proteins and potential therapeutic targets.
our Copper(I)-Nitrene Platform (CuNiP), particularly in comparison to the work by Chang et al.Indeed, the issue of competitive sulfoxidation is a prevalent challenge in the oxidative modification of methionine, as also observed in the oxaziridine probe(Chang et.al., Science  2017, 355, 597-602, Fig. 2; J. Am.Chem.Soc.2019,141, 12657-12662, supporting information, Page S7).The primary objective of our work is to develop new, mechanistically and structurally different labelling strategies for methionine and apply them for profiling of hypereactive methionine.Till date, excellent reactivity and chemoselectivity of nitrenes have not been harnessed for protein bioconjugation and profiling.We wanted to utilize this concept in chemoselective labeling of methionine.In this context, we have made the following advancements:1.Discovery of New Protein labeled sites: